I have been doing research on gelatin microparticles (crosslinked glutaraldehyde), and I need to perform qualitative analysis by using a confocal fluorescence microscope. However, the autofluorescence (AF) of gelatin is disturbing. It exhibited a high signal and hindered all target areas that I wanted to check.
What I have done to solve this:
1. Wash with glycine many times -> not working
2. Perform chemical bleaching with Sudan Black; the chemical is well-known to bleach the tissue, condition is 0.1%w/v, 1hr incubation at RT -> not working
3. Perform photo bleaching with UV and LED lamps; Low intensity, more than 24 hour exposure -> not working
4. My friend suggested me to do the images post-processing -> still I cannot get a reliable image from that.
I have been also discussed with the operator of confocal microscope.
She said it need a pulsed-laser and time-gating function for confocal microscope.
But I don't have that in my institute.
Detail: Gelatin has AF around 400-600nm ex/em so it covers all red and green color signals. So, it remains only blue color zone which will be hard for me because I need to check around 5 proteins there.
So, is there any other method to reduce the autofluorescence of gelatin microparticles?
Hi Nopphado
Sorry, but "perform a qualitative analysis", of what do you want to perform? What is the useful signal that is being affected by the AF? Please clarify
Lëonid Volkov
Thanks for your attention in my question.
The qualitative analysis in my terms is the image result.
For example, I want to check Live/Dead assay by using calcein and Ethd-1 staining. It will appear green and red color as a live and dead cell when checking with fluorescence microscope. However both green and red color was affected by AF of gelatin. In this case, I can use WST assay to check viability but that is quantitative analysis.
The example is just one part. My research is dealing with many protein binding so I will do a lot of immunostaining such as vinculin-AlexFlour488, paxilin-FITC, etc. which will express green or red color.
Some said why don't I use blue color instead, yes, it's possible. But my work will be overloaded especially the sample preparation.
Hi Nopphado
From my point of view, the best solution in your case would be to use dyes with large stokes shift, such as StarBright Violet 515; StarBright Violet 610 and so on.
Best of luck
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